Project Summary/Abstract Asymmetrical localization and specific protein binding patterns of mRNA play a key role in many cellular pro- cesses, and aberrant mRNA localization has been observed in cancer and several neurological diseases. Gain- ing a deeper understanding of mRNA localization patterns and the corresponding mechanisms of mRNA transport would provide valuable information regarding disease progression and potential therapeutic ap- proaches. However, the RNA labeling methods currently available suffer from various challenges and limitations, creating an ongoing demand for improved methods for labeling and imaging of specific mRNA sequences in living cells. Harnessing the power of molecular recognition between RNA and small molecules, we have devel- oped ribozyme sequences that are capable of self-alkylation with an electrophilic fluorescein analogue, and can be fused to an mRNA of interest and expressed in cells. These ribozymes are anticipated to serve the dual purpose of enabling fluorescence-based visualization of mRNA and providing a handle for immunoprecipitation of proteins bound to specific RNA sequences. Compared with other RNA labeling technologies, the proposed ribozyme-based approach offers the benefits of smaller fusion size, potential for use with a broad palette of small-molecule fluorophores, and reduction of background signal by removal of excess fluorophore. Additionally, the proposed ribozyme-based approach enables new applications that are not possible with other RNA labeling technologies, such as pulse-chase labeling and transcript-specific immunoprecipitation. The specific aims of this project are to: (1) utilize the ribozymes to isolate and identify transcript-specific RNA-binding proteins; (2) fluorescently label and visualize mRNAs in living cells and monitor time-resolved mRNA dynamics; (3) utilize our RNA labeling and immunoprecipitation methods to gain insight into the mechanism and specific localization pat- terns of non-canonical ER-localized mRNAs. This research is anticipated to provide powerful tools for studying the localization and transport mechanisms of mRNA in living cells, and will put these tools to immediate use to answer biological questions regarding ER-localized RNAs. This is in turn expected to further our understanding of fundamental cellular processes and offer new insights into the mechanisms and treatment of disease.